The Beta-adrenergic receptor kinase (beta-ARK) phosphorylates a number of G protein-coupled receptors in an agonist-dependent manner. Beta-ARK serves as a model for the family of signal-dependent kinases which play a role in adaptation of a call to continuous stimulation. Desensitization to hormones and drugs which activate G protein-coupled receptors diminish the effectiveness of a variety of agents used in the treatment of asthma congestive heart failure, and hypertension. Information concerning the structural requirements of the kinase and its substrates will lead to the development of novel inhibitors of beta-ARK mediated desensitization. Such inhibitors may be clinically useful as therapeutic agents for the treatment of these diseases. The long-term goal of this proposal is to define the structural features which contribute to the signal-dependent phosphorylation reaction. Three specific aims are as follows: (1) Identify the sites of beta-ARK phosphorylation present in the beta2-adrenergic receptor. (2) Determine the mechanism of signal-dependent phosphorylation by beta-ARK. (3) Identify regions of beta-ARK and the beta2-adrenergic receptor which contribute to their interaction. With the use of the baculovirus expression system, unprecedented amounts of kinase and receptor are available for biochemical studies. HPLC techniques specifically designed to provide high recovery will be used to obtain phosphopeptides for direct sequencing and mapping studies. Kinetic studies with peptides and receptor substrates will be used to characterize mechanisms of the signal-dependent phosphorylation reaction. Finally, a combination of limited proteolysis, group-specific modification of key amino acids, site-directed mutagenesis and label transfer experiments will serve to identify regions involved in beta-ARK/beta2-adrenergic receptor interactions.